国际生殖健康/计划生育杂志

国际生殖健康/计划生育 ›› 2014, Vol. 33 ›› Issue (2): 95-98.

• 论著 • 上一篇    下一篇

小鼠Tox3基因重组慢病毒载体的构建及其在卵巢颗粒细胞中的表达


崔玉倩，赵涵，马增香，赵世斗，徐海静，王文婷，郭海鹏，陈子江   

  1. 200127 上海交通大学医学院附属仁济医院生殖医学中心
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2014-03-15 发布日期:2014-03-15
  • 通讯作者: 陈子江

Construction of the Recombinant Lentiviral Vector Containing Mouse Tox3 Gene and Its Expression in Primary Granulosa Cells

CUI Yu-qian，ZHAO Han，MA Zeng-xiang，ZHAO Shi-dou，XU Hai-jing，WANG Wen-ting，GUO Hai-peng，CHEN Zi-jiang   

  1. Reproductive Medical Center，Renji Hospital，Shanghai Jiao Tong University School of Medicine，Shanghai 200127，China
  • Received:1900-01-01 Revised:1900-01-01 Published:2014-03-15 Online:2014-03-15
  • Contact: CHEN Zi-jiang

PDF (PC)

7447

摘要： 目的：构建重组慢病毒载体pCDH-Tox3-Flag，并检测其转染小鼠原代卵泡颗粒细胞后的表达情况。方法：利用DNA重组技术将小鼠Tox3基因克隆入慢病毒表达载体pCDH-MCS-T2A-copGFP-MSCV中，通过酶切、测序验证后，将重组慢病毒载体pCDH-Tox3-Flag、包装质粒psPAX2和包膜质粒pMD2.G共转染HEK293FT细胞，包装重组慢病毒LV-Tox3-Flag，并感染小鼠原代颗粒细胞，用反转录聚合酶链反应（RT-PCR）检测Tox3 mRNA的转录水平，蛋白质印迹（Western blot）法检测TOX3-Flag蛋白的表达情况。结果：经酶切及测序结果证实，构建了重组慢病毒载体LV-Tox3；RT-PCR及Western blot 结果显示慢病毒感染小鼠原代颗粒细胞后，Tox3基因能在细胞内正确转录、翻译并稳定表达TOX3蛋白。结论：成功建立Tox3基因慢病毒表达载体LV-Tox3-Flag，包装的慢病毒能够成功感染小鼠原代颗粒细胞，并使Tox3基因得到稳定表达，为后续研究Tox3基因在生殖相关疾病的生理与病理作用奠定了基础。

关键词: Tox3, 慢病毒载体, 粒层细胞, 小鼠

Abstract: Objective: To construct the recombinant lentiviral vector containing mouse Tox3 gene, and to check its expression in primary granulosa cells. Methods: The full length of Tox3 fragment was amplified by PCR, and subcloned into the lentiviral vector pCDH-MCS-T2A-copGFP-MSCV. The resultant lentivirus were confirmed by PCR, restriction enzyme digestion and DNA sequencing． The recombinant lentivirus （LV-Tox3-Flag） were produced from HEK293FT by a transient co-transfection with psPAX2 and pMD2.G. Primary granulosa cells were infected by LV-Tox3-Flag lentivirus，and the expression of TOX3 was confirmed by RT-PCR and Western blot. Results: The recombinant lentiviral vector carried the Tox3 gene was successfully constructed. RT-PCR and Western blot analysis revealed that the Tox3 gene can be correctly transcript and translated in the granulosa cells infected by LV-Tox3. Conclusions: The recombinant LV-Tox3-Flag was successfully constructed. It will be used to transfect target cells in our future study．

Key words: Tox3, Lentiviral vector, Granulosa cells, Mice